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Title: Tunneling nanotubes mediate the expression of senescence markers in mesenchymal stem/stromal cell spheroids
Abstract

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is limited by acquired senescence following prolonged culture expansion and high-passage numbers. However, the degree of cell senescence is dynamic, and cell-cell communication is critical to promote cell survival. MSC spheroids exhibit improved viability compared with monodispersed cells, and actin-rich tunneling nanotubes (TNTs) may mediate cell survival and other functions through the exchange of cytoplasmic components. Building upon our previous demonstration of TNTs bridging MSCs within these cell aggregates, we hypothesized that TNTs would influence the expression of senescence markers in MSC spheroids. We confirmed the existence of functional TNTs in MSC spheroids formed from low-passage, high-passage, and mixtures of low- and high-passage cells using scanning electron microscopy, confocal microscopy, and flow cytometry. The contribution of TNTs toward the expression of senescence markers was investigated by blocking TNT formation with cytochalasin D (CytoD), an inhibitor of actin polymerization. CytoD-treated spheroids exhibited decreases in cytosol transfer. Compared with spheroids formed solely of high-passage MSCs, the addition of low-passage MSCs reduced p16 expression, a known genetic marker of senescence. We observed a significant increase in p16 expression in high-passage cells when TNT formation was inhibited, establishing the importance of TNTs in MSC spheroids. These data confirm the restorative role of TNTs within MSC spheroids formed with low- and high-passage cells and represent an exciting approach to use higher-passage cells in cell-based therapies.

</sec> </span> <a href='#' class='show open-abstract' style='margin-left:10px;'>more »</a> <a href='#' class='hide close-abstract' style='margin-left:10px;'>« less</a> <div style="clear:both;margin-bottom:20px;"></div> <dl class="dl-horizontal small"> <dt>NSF-PAR ID:</dt> <dd>10363126</dd> </dl> <dl class="dl-horizontal small"> <dt>Author(s) / Creator(s):</dt> <dd> <a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Whitehead, Jacklyn""><span class="author" itemprop="author">Whitehead, Jacklyn</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Zhang, Jiali""><span class="author" itemprop="author">Zhang, Jiali</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Harvestine, Jenna N.""><span class="author" itemprop="author">Harvestine, Jenna N.</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Kothambawala, Alefia""><span class="author" itemprop="author">Kothambawala, Alefia</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Liu, Gang-yu""><span class="author" itemprop="author">Liu, Gang-yu</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Leach, J. Kent""><span class="author" itemprop="author">Leach, J. Kent</span> <sup class="text-muted"></sup></a></dd> </dl> <dl class="dl-horizontal small"> <dt>Publisher / Repository:</dt> <dd itemprop="publisher">Oxford University Press</dd> </dl> <dl class="dl-horizontal small"> <dt>Date Published:</dt> <dd> <time itemprop="datePublished" datetime="2019-08-01">2019-08-01</time> </dd> </dl> <dl class="dl-horizontal small"> <dt>Journal Name:</dt> <dd>Stem Cells</dd> </dl> <dl class="dl-horizontal small"> <dt>Volume:</dt> <dd>38</dd> </dl> <dl class="dl-horizontal small"> <dt>Issue:</dt> <dd>1</dd> </dl> <dl class="dl-horizontal small"> <dt>ISSN:</dt> <dd>1066-5099</dd> </dl> <dl class="dl-horizontal small"> <dt>Page Range / eLocation ID:</dt> <dd>p. 80-89</dd> </dl> <dl class="dl-horizontal small"> <dt>Format(s):</dt> <dd>Medium: X</dd> </dl> <dl class="dl-horizontal small semi-colon-delimited-data"> <dt>Sponsoring Org:</dt> <dd itemprop="sourceOrganization"> <span>National Science Foundation</span> </dd> </dl> <div class="clearfix"></div> </div> </div> <div id="citation-addl" class="hidden-print"> <h5 id='mlt-header'>More Like this</h5> <ol class="item-list documents" id="citation-mlt" style="min-height: 80px;"> <li> <div class="article item document" itemscope itemtype="http://schema.org/TechArticle"> <div class="item-info"> <div class="title"> <a href="https://par.nsf.gov/biblio/10337868-tunneling-nanotubes-between-cells-migrating-ecm-mimicking-fibrous-environments" itemprop="url"> <span class='span-link' itemprop="name">Tunneling Nanotubes between Cells Migrating in ECM Mimicking Fibrous Environments</span> </a> </div> <div> <strong> <a class="misc external-link" href="https://doi.org/10.3390/cancers14081989" target="_blank" title="Link to document DOI">https://doi.org/10.3390/cancers14081989  <span class="fas fa-external-link-alt"></span></a> </strong> </div> <div class="metadata"> <span class="authors"> <span class="author" itemprop="author">Jana, Aniket</span> <span class="sep">; </span><span class="author" itemprop="author">Ladner, Katherine</span> <span class="sep">; </span><span class="author" itemprop="author">Lou, Emil</span> <span class="sep">; </span><span class="author" itemprop="author">Nain, Amrinder S.</span> </span> <span class="year">( <time itemprop="datePublished" datetime="2022-04-01">April 2022</time> , Cancers) </span> </div> <div style="cursor: pointer;-webkit-line-clamp: 5;" class="abstract" itemprop="description"> Tunneling nanotubes (TNTs) comprise a unique class of actin-rich nanoscale membranous protrusions. They enable long-distance intercellular communication and may play an integral role in tumor formation, progression, and drug resistance. TNTs are three-dimensional, but nearly all studies have investigated them using two-dimensional cell culture models. Here, we applied a unique 3D culture platform consisting of crosshatched and aligned fibers to fabricate synthetic suspended scaffolds that mimic the native fibrillar architecture of tumoral extracellular matrix (ECM) to characterize TNT formation and function in its native state. TNTs are upregulated in malignant mesothelioma; we used this model to analyze the biophysical properties of TNTs in this 3D setting, including cell migration in relation to TNT dynamics, rate of TNT-mediated intercellular transport of cargo, and conformation of TNT-forming cells. We found that highly migratory elongated cells on aligned fibers formed significantly longer but fewer TNTs than uniformly spread cells on crossing fibers. 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In sum, our strategy for culturing cells in ECM-mimicking bioengineered scaffolds provides a new approach for accurate biophysical and biologic assessment of TNT formation and structure in native fibrous microenvironments. </div> <a href='#' class='show open-abstract' style='margin-left:10px;'>more »</a> <a href='#' class='hide close-abstract' style='margin-left:10px;'>« less</a> </div><div class="clearfix"></div> </div> </li> <li> <div class="article item document" itemscope itemtype="http://schema.org/TechArticle"> <div class="item-info"> <div class="title"> <a href="https://par.nsf.gov/biblio/10460320-morphogen-delivery-osteoconductive-nanoparticles-instructs-stromal-cell-spheroid-phenotype" itemprop="url"> <span class='span-link' itemprop="name">Morphogen Delivery by Osteoconductive Nanoparticles Instructs Stromal Cell Spheroid Phenotype</span> </a> </div> <div> <strong> <a class="misc external-link" href="https://doi.org/10.1002/adbi.201900141" target="_blank" title="Link to document DOI">https://doi.org/10.1002/adbi.201900141  <span class="fas fa-external-link-alt"></span></a> </strong> </div> <div class="metadata"> <span class="authors"> <span class="author" itemprop="author">Whitehead, Jacklyn</span> <span class="sep">; </span><span class="author" itemprop="author">Kothambawala, Alefia</span> <span class="sep">; </span><span class="author" itemprop="author">Kent Leach, J.</span> </span> <span class="year">( <time itemprop="datePublished" datetime="2019-10-01">October 2019</time> , Advanced Biosystems) </span> </div> <div style="cursor: pointer;-webkit-line-clamp: 5;" class="abstract" itemprop="description"> <title>Abstract

Mesenchymal stem/stromal cells (MSCs) exhibit a rapid loss in osteogenic phenotype upon removal of osteoinductive cues, as commonly occurs during transplantation. Osteogenic differentiation can be more effectively but not fully maintained by aggregating MSCs into spheroids. Therefore, the development of effective strategies that prolong the efficacy of inductive growth factors would be advantageous for advancing cell‐based therapies. To address this challenge, osteoinductive bone morphogenetic protein‐2 (BMP‐2) is adsorbed to osteoconductive hydroxyapatite (HA) nanoparticles for incorporation into MSC spheroids. MSC induction is evaluated in osteogenic conditions and retention of the osteogenic phenotype in the absence of other osteogenic cues. HA is more uniformly incorporated into spheroids at lower concentrations, while BMP‐2 dosage is dependent upon initial morphogen concentration. MSC spheroids containing BMP‐2‐loaded HA nanoparticles exhibit greater alkaline phosphatase activity and more uniform spatial expression of osteocalcin compared to spheroids with uncoated HA nanoparticles. Spheroids cultured in media containing soluble BMP‐2 demonstrate differentiation only at the spheroid periphery. Furthermore, the osteogenic phenotype of MSC spheroids is better retained with BMP‐2‐laden HA upon the removal of soluble osteogenic cues. These findings represent a promising strategy for simultaneous delivery of osteoconductive and osteoinductive signals for enhancing MSC participation in bone formation.

 
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  • Abstract

    Human cerebral organoids derived from induced pluripotent stem cells (iPSCs) provide novel tools for recapitulating the cytoarchitecture of human brain and for studying biological mechanisms of neurological disorders. However, the heterotypic interactions of neurovascular units, composed of neurons, pericytes, astrocytes, and brain microvascular endothelial cells, in brain-like tissues are less investigated. The objective of this study is to investigate the impacts of neural spheroids and vascular spheroids interactions on the regional brain-like tissue patterning in cortical spheroids derived from human iPSCs. Hybrid neurovascular spheroids were constructed by fusion of human iPSC-derived cortical neural progenitor cell (iNPC) spheroids, endothelial cell (iEC) spheroids, and the supporting human mesenchymal stem cells (MSCs). Single hybrid spheroids were constructed at different iNPC: iEC: MSC ratios of 4:2:0, 3:2:1 2:2:2, and 1:2:3 in low-attachment 96-well plates. The incorporation of MSCs upregulated the secretion levels of cytokines VEGF-A, PGE2, and TGF-β1 in hybrid spheroid system. In addition, tri-cultured spheroids had high levels of TBR1 (deep cortical layer VI) and Nkx2.1 (ventral cells), and matrix remodeling genes, MMP2 and MMP3, as well as Notch-1, indicating the crucial role of matrix remodeling and cell-cell communications on cortical spheroid and organoid patterning. Moreover, tri-culture system elevated blood-brain barrier gene expression (e.g., GLUT-1), CD31, and tight junction protein ZO1 expression. Treatment with AMD3100, a CXCR4 antagonist, showed the immobilization of MSCs during spheroid fusion, indicating a CXCR4-dependent manner of hMSC migration and homing. This forebrain-like model has potential applications in understanding heterotypic cell-cell interactions and novel drug screening in diseased human brain.

     
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  • Abstract

    Collagen is the major structural protein in myocardium and contributes to tissue strength and integrity, cellular orientation, and cell–cell and cell‐matrix interactions. Significant post‐myocardial infarction related loss of cardiomyocytes and cardiac tissue, and their subsequent replacement with fibrous scar tissue, negatively impacts endogenous tissue repair and regeneration capabilities. To overcome such limitations, tissue engineers are working toward developing a 3D cardiac patch which not only mimics the structural, functional, and biological hierarchy of the native cardiac tissue, but also could deliver autologous stem cells and encourage their homing and differentiation. In this study, we examined the utility of electrospun, randomly‐oriented, type‐I collagen nanofiber (dia= 789 ± 162 nm) mats on the cardiomyogenic differentiation of human bone marrow‐derived mesenchymal stem cells (BM‐MSC) spheroids, in the presence or absence of 10 μM 5‐azacytidine (aza). Results showed that these scaffolds are biocompatible and enable time‐dependent evolution of early (GATA binding protein 4: GATA4), late (cardiac troponin I: cTnI), and mature (myosin heavy chain: MHC) cardiomyogenic markers, with a simultaneous reduction in CD90 (stemness) expression, independent of aza‐treatment. Aza‐exposure improved connexin‐4 expression and sustained sarcomeric α‐actin expression, but provided only transient improvement in cardiac troponin T (cTnT) expression. Cell orientation and alignment significantly improved in these nanofiber scaffolds over time and with aza‐exposure. Although further quantitativein vitroandin vivostudies are needed to establish the clinical applicability of such stem‐cell laden collagen nanofiber mats as cardiac patches for cardiac tissue regeneration, our results underscore the benefits of 3D milieu provided by electrospun collagen nanofiber mats, aza, and spheroids on the survival, cardiac differentiation and maturation of human BM‐MSCs. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3303–3312, 2018.

     
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  • Abstract

    In vivo mesenchymal stem cell (MSC) survival is relevant to therapeutic applications requiring engraftment and potentially to nonengraftment applications as well. MSCs are a mixture of progenitors at different stages of cellular aging, but the contribution of this heterogeneity to the survival of MSC implants is unknown. Here, we employ a biomarker of cellular aging, the decoy TRAIL receptor CD264, to compare the survival kinetics of two cell populations in human bone marrow MSC (hBM‐MSC) cultures. Sorted CD264+hBM‐MSCs from two age‐matched donors have elevated β‐galactosidase activity, decreased differentiation potential and form in vitro colonies inefficiently relative to CD264hBM‐MSCs. Counterintuitive to their aging phenotype, CD264+hBM‐MSCs exhibited comparable survival to matched CD264hBM‐MSCs from the same culture during in vitro colony formation and in vivo when implanted ectopically in immunodeficient NIH III mice. In vitro and in vivo survival of these two cell populations were independent of colony‐forming efficiency. These findings have ramifications for the preparation of hBM‐MSC therapies given the prevalence of aging CD264+cells in hBM‐MSC cultures and the popularity of colony‐forming efficiency as a quality control metric in preclinical and clinical studies with MSCs.

     
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